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Luciferase assay reagents containing luciferin are inherently limited by the conflict between a substrate that is activated with increasing pH and reagent components that are more stable with decreasing pH. Addition of a fluorine at the 5’-position of luciferin allows for activation of the substrate at a lower pH, and this provides a number of advantages in a luciferase reagent. The oxidation and/or degradation of luciferase reagent components is less at lower pH, so 5’- fluoroluciferin-containing reagents are more stable than those containing luciferin. Luciferase is also less affected by enzymatic inhibitors in a low pH reagent and the color quenching of phenol red, a dye commonly found in cell culture media, is reduced. Furthermore, low pH leads to a greatly reduced thiol odor; luciferin-containing reagents typically require high concentrations of thiols to control the rate of enzymatic turnover and generate a steady signal, but the low pH of reagents containing 5’-fluoroluciferin can reduce the rate of turnover using 100-fold less thiol. Thus, the use of 5’-fluoroluciferin allows for a luciferase assay reagent that is easier to use, more robust to reaction environment, and less aromatic than reagents containing unmodified luciferin.

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